The inflammatory response of lymphatic endothelium

Lymphatic vessels have traditionally been regarded as a rather inert drainage system, which just passively transports fluids, leukocytes and antigen. However, it is becoming increasingly clear that the lymphatic vasculature is highly dynamic and plays a much more active role in inflammatory and immune processes. Tissue inflammation induces a rapid, stimulus-specific upregulation of chemokines and adhesion molecules in lymphatic endothelial cells and a proliferative expansion of the lymphatic network in the inflamed tissue and in draining lymph nodes. Moreover, increasing evidence suggests that inflammation-induced changes in the lymphatic vasculature have a profound impact on the course of inflammatory and immune responses, by modulating fluid drainage, leukocyte migration or the removal of inflammatory mediators from tissues. In this review we will summarize and discuss current knowledge of the inflammatory response of lymphatic endothelium and of inflammation-induced lymphangiogenesis and the current perspective on the overall functional significance of these processes.     

Role of bone marrow-derived lymphatic endothelial progenitor cells for lymphatic neovascularization

The lymphatic vasculature plays a pivotal role in maintaining tissue fluid homeostasis, immune surveillance, and lipid uptake in the gastrointestinal organs. Therefore, impaired function of the lymphatic vessels caused by genetic defects, infection, trauma, or surgery leads to the abnormal accrual of lymph fluid in the tissue and culminates in the swelling of affected tissues, known as lymphedema. Lymphedema causes impaired wound healing, compromised immune defense, and, in rare case, lymphangiosarcoma. Although millions of people suffer from lymphedema worldwide, no effective therapy is currently available. In addition, recent advances in cancer biology have disclosed an indispensable function of the lymphatic vessel in tumor growth and metastasis. Therefore, understanding the detailed mechanisms governing lymphatic vessel formation and function in pathophysiologic conditions is essential to prevent or treat these diseases. We review the developmental processes of the lymphatic vessels and postnatal lymphatic neovascularization, focusing on the role of recently identified bone marrow-derived podoplanin-expressing (podoplanin(+)) cells as lymphatic endothelial progenitor cells.     

Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling

Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.     

Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-β signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct''s potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

The lymphatic and blood vascular systems are two distinct vessel network systems that work in synchrony to maintain tissue homeostasis. Blood vessels transport oxygen and nutrients around the body, while lymphatic vessels collect leaked fluid and macromolecules from the interstitial space and return them to the blood circulation, maintaining interstitial fluid homeostasis (1). Furthermore, the lymphatic system plays a central role in immune responses, inflammation regulation, and lipid absorption (2). While many in vitro models have been created to study angiogenesis, fewer attempts have been made to engineer an in vitro platform to study lymphangiogenesis. Such engineered models are critical for both fundamental research and the development of clinically implantable tissue to treat various diseases involving the lymphatic system. One such disease is lymphedema, a chronic condition that affects 200 million people worldwide (3). Lymphedema is characterized by tissue swelling resulting from a compromised lymphatic system. The condition is mainly caused by complications during cancer treatment but may also develop due to genetic disorders. The condition is progressive and incurable, with a high risk of infection. Implantation of engineered lymphatic tissue can serve as a treatment for such disease (4).Lymph flow is primarily driven by pressures generated by lymphatic contractions of the smooth muscle cells surrounding the vessels (5). Impaired contractility thus reduces lymph flow and may cause lymphedema. Previous computational studies have investigated the correlation between lymphatic vessel contractility and mechanical stimulation, such as mechanical loading, pressure gradients, and shear stress amplitudes (6, 7). Furthermore, studies have investigated lymphatic vessel capacity to distend under mechanical loading conditions. In addition, the microenvironment composition has been shown to play an important role in enabling lymphatic vessel functionality (4).Thus far, several groups have been able to engineer lymphatic tissues. Marino et al. created dermo-epidermal skin grafts with lymphatic and blood vessels embedded in a fibrin-collagen gel (8). Others created a lymphatic vessel network within multilayered fibroblast sheets (9, 10). Another study demonstrated that different hydrogel compositions are required for the optimal growth and development of blood and lymphatic endothelial cells (BECs and LECs, respectively) (11). However, no studies have investigated the influence of the supporting cells, the secreted extracellular matrix (ECM), and the mechanical environment on the forming lymphatic vessels. Since lymphatic pathologies are known to correlate with mechanically impaired lymphatic vessels (4), it is important to create lymphatic models with a biomimetic microenvironment.In this study, lymphatic vessel networks were engineered to investigate fundamental questions concerning lymphangiogenesis, including the influence of different supporting cells on the formation of lymphatic vessels and the role of PDGFR-β, an important receptor associated with support cells recruitment, in vessel formation. In addition, a complex tissue designed to better mimic native tissue was generated and lymphatic and blood vessel development along with muscle formation were monitored. In addition, the impact of the application of cyclic stretch on the organization and alignment of lymphatic-blood-vessel-muscle tissue was assessed. Finally, the penetration and anastomosis of the engineered lymphatic vessels were monitored following their implantation into mice.     

Sphingosine-1-phosphate promotes lymphangiogenesis by stimulating S1P1/Gi/PLC/Ca2+ signaling pathways

The lymphatic system plays pivotal roles in mediating tissue fluid homeostasis and immunity, and excessive lymphatic vessel formation is implicated in many pathological conditions, which include inflammation and tumor metastasis. However, the molecular mechanisms that regulate lymphatic vessel formation remain poorly characterized. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that is implicated in a variety of biologic processes such as inflammatory responses and angiogenesis. Here, we first report that S1P acts as a lymphangiogenic mediator. S1P induced migration, capillary-like tube formation, and intracellular Ca(2+) mobilization, but not proliferation, in human lymphatic endothelial cells (HLECs) in vitro. Moreover, a Matrigel plug assay demonstrated that S1P promoted the outgrowth of new lymphatic vessels in vivo. HLECs expressed S1P1 and S1P3, and both RNA interference-mediated down-regulation of S1P1 and an S1P1 antagonist significantly blocked S1P-mediated lymphangiogenesis. Furthermore, pertussis toxin, U73122, and BAPTA-AM efficiently blocked S1P-induced in vitro lymphangiogenesis and intracellular Ca(2+) mobilization of HLECs, indicating that S1P promotes lymphangiogenesis by stimulating S1P1/G(i)/phospholipase C/Ca(2+) signaling pathways. Our results suggest that S1P is the first lymphangiogenic bioactive lipid to be identified, and that S1P and its receptors might serve as new therapeutic targets against inflammatory diseases and lymphatic metastasis in tumors.     

Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.     

Impaired lymphatic contraction associated with immunosuppression

To trigger an effective immune response, antigen and antigen-presenting cells travel to the lymph nodes via collecting lymphatic vessels. However, our understanding of the regulation of collecting lymphatic vessel function and lymph transport is limited. To dissect the molecular control of lymphatic function, we developed a unique mouse model that allows intravital imaging of autonomous lymphatic vessel contraction. Using this method, we demonstrated that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions. By contrast, under inflammatory conditions, inducible NOS (iNOS)-expressing CD11b(+)Gr-1(+) cells attenuate lymphatic contraction. This inhibition of lymphatic contraction was associated with a reduction in the response to antigen in a model of immune-induced multiple sclerosis. These results suggest the suppression of lymphatic function by the CD11b(+)Gr-1(+) cells as a potential mechanism of self-protection from autoreactive responses during on-going inflammation. The central role for nitric oxide also suggests that other diseases such as cancer and infection may also mediate lymphatic contraction and thus immune response. Our unique method allows the study of lymphatic function and its molecular regulation during inflammation, lymphedema, and lymphatic metastasis.     

Control of dendritic cell trafficking in lymphatics by chemokines

Dendritic cells (DCs) are crucial participants in maintaining immune surveillance of the periphery and initiating primary immune responses within the draining lymph nodes. The afferent lymphatic vessels provide a conduit for this essential trafficking and, as this review will describe, play an active role in regulating DC migration. Afferent lymphatic capillaries support constitutive trafficking of DCs from resting, non-inflamed tissue, to maintain tolerance against self-antigen and to provide immune surveillance. Following exposure to pathogens or pro-inflammatory cytokines, DCs mature from phagocytes to professional antigen-presenting cells, whilst the lymphatic endothelium adopts an activated phenotype to support the ensuing increase in leukocyte trafficking. The lymphatic endothelial-derived chemokine CCL21 plays a well-characterized role in directing migration of CCR7⁺ DC in both resting and acute inflammatory conditions. However, efficient trafficking of DCs from inflamed tissue also demands additional chemokine-receptor pairs. Thus, entry of DCs to activated lymphatic vessels is an intricately regulated multi-step process involving numerous chemokines and adhesion molecules.     

Interstitial flow as a guide for lymphangiogenesis

The lymphatic system is important in tissue fluid balance regulation, immune cell trafficking, edema, and cancer metastasis, yet very little is known about the sequence of events that initiate and coordinate lymphangiogenesis. Here, we characterize the process of lymphatic regeneration by uniquely correlating interstitial fluid flow and lymphatic endothelial cell migration with lymphatic function. A new model of skin regeneration using a collagen implant in a mouse tail has been developed, and it shows that (1) interstitial fluid channels form before lymphatic endothelial cell organization and (2) lymphatic cell migration, vascular endothelial growth factor-C expression, and lymphatic capillary network organization are initiated primarily in the direction of lymph flow. These data suggest that interstitial fluid channeling precedes and may even direct lymphangiogenesis (in contrast to blood angiogenesis, in which fluid flow proceeds only after the vessel develops); thus, a novel and robust model is introduced for correlating molecular events with functionality in lymphangiogenesis.     

Angiopoietin-1 promotes lymphatic sprouting and hyperplasia

Angiopoietin 1 (Ang1), a ligand for the receptor tyrosine kinase Tie2, regulates the formation and stabilization of the blood vessel network during embryogenesis. In adults, Ang1 is associated with blood vessel stabilization and recruitment of perivascular cells, whereas Ang2 acts to counter these actions. Recent results from gene-targeted mice have shown that Ang2 is also essential for the proper patterning of lymphatic vessels and that Ang1 can be substituted for this function. In order to characterize the effects of the angiopoietins on lymphatic vessels, we employed viral vectors for overexpression of Ang1 in adult mouse tissues. We found that Ang1 activated lymphatic vessel endothelial proliferation, vessel enlargement, and generation of long endothelial cell filopodia that eventually fused, leading to new sprouts and vessel development. Cutaneous lymphatic hyperplasia was also detected in transgenic mice expressing Ang1 in the basal epidermal cells. Tie2 was expressed in the lymphatic endothelial cells and Ang1 stimulation of these cells resulted in up-regulation of vascular endothelial growth factor receptor 3 (VEGFR-3). Furthermore, a soluble form of VEGFR-3 inhibited the observed lymphatic sprouting. Our results reinforce the concept that Ang1 therapy may be useful in settings of tissue edema.     

Dynamics of airway blood vessels and lymphatics: lessons from development and inflammation

Blood vessels and lymphatic vessels in the respiratory tract play key roles in inflammation. By undergoing adaptive remodeling and growth, blood vessels undergo changes that enable the extravasation of plasma and leukocytes into inflamed tissues, and lymphatic vessels adjust to the increased fluid clearance and cell traffic involved in immune responses. Blood vessels and lymphatics in adult airways are strikingly different from those of late-stage embryos. Before birth, blood vessels in mouse airways make up a primitive plexus similar to that of the yolk sac. This plexus undergoes rapid and extensive remodeling at birth. In the early neonatal period, parts of the plexus regress. Capillaries then rapidly regrow, and with arterioles and venules form the characteristic adult vascular pattern. Lymphatic vessels of the airways also undergo rapid changes around birth, when lymphatic endothelial cells develop button-like intercellular junctions specialized for efficient fluid uptake. Among the mechanisms that underlie the onset of rapid vascular remodeling at birth, changes in tissue oxygen tension and mechanical forces associated with breathing are likely to be involved, along with growth factors that promote the growth and maturation of blood vessels and lymphatics. Whatever the mechanisms, the dynamic nature of airway blood vessels and lymphatics during perinatal development foretells the extraordinary vascular plasticity found in many diseases.     

Hepatocyte growth factor is a lymphangiogenic factor with an indirect mechanism of action

Hepatocyte growth factor (HGF) has previously been reported to act as a hemangiogenic factor, as well as a mitogenic factor for a variety of tumor cells. Here, we demonstrate that HGF is a lymphangiogenic factor, which may contribute to lymphatic metastasis when overexpressed in tumors. In a mouse corneal lymphangiogenesis model, implantation of HGF induces sprouting and growth of new lymphatic vessel expressing the lymphatic vessel endothelial specific marker hyaluronan receptor-1 (Lyve-1). Unlike blood vessels, the Lyve-1-positive structures consist of blunt-ended vessels of large diameters that generally lack expression of CD31. The growth of HGF-induced lymphatic vessels can be partially blocked by a soluble VEGFR-3, suggesting that HGF may stimulate lymphatic vessel growth through an indirect mechanism. Consistent with this finding, the HGF receptor (c-Met) is only localized on corneal blood vessels but is absent on lymphatic vessels in a mouse corneal assay. In a transgenic mouse model that expresses HGF under the control of the whey acidic protein (WAP) gene promoter, transgenic females develop tumors in the mammary glands after several pregnancies. Interestingly, dilated Lyve-1-positive lymphatic vessels accumulate in the peritumoral area and occasionally penetrate into the tumor tissue. Our findings indicate that HGF may play a critical role in lymphangiogenesis and potentially contribute to lymphatic metastasis.     

淋巴管:干预动脉粥样硬化发生发展的潜在途径

动脉粥样硬化是一种慢性炎症性疾病,是血管壁对各种损伤的异常反应。虽然影响动脉粥样硬化的因素很多,但淋巴管在动脉粥样硬化中的作用一直被忽视。传统上认为淋巴管是将间质液回流至血液循环的通道。在早期的研究中,发现动脉粥样硬化周围存在大量淋巴管,但两者之间的关系一直不清楚。近期研究发现淋巴管不仅参与动脉炎症的起始和消退,在胆固醇逆转运中也发挥着积极作用。此外,改善淋巴功能或促进局部淋巴管生成似乎可以减轻动脉粥样硬化的进展。因此,研究淋巴管与动脉粥样硬化的关系对干预动脉粥样硬化的发生发展具有重要意义。文章介绍了淋巴管与动脉粥样硬化发生发展相关的炎症、胆固醇逆转运以及免疫等因素的关系,以期为动脉粥样硬化干预策略的研究提供新的视角。     

Mast cell modulation of the vascular and lymphatic endothelium

Mast cells (MCs) promote a wide range of localized and systemic inflammatory responses. Their involvement in immediate as well as chronic inflammatory reactions at both local and distal sites points to an extraordinarily powerful immunoregulatory capacity with spatial and temporal versatility. MCs are preferentially found in close proximity to both vascular and lymphatic vessels. On activation, they undergo a biphasic secretory response involving the rapid release of prestored vasoactive mediators followed by de novo synthesized products. Many actions of MCs are related to their capacity to regulate vascular flow and permeability and to the recruitment of various inflammatory cells from the vasculature into inflammatory sites. These mediators often work in an additive fashion and achieve their inflammatory effects locally by directly acting on the vascular and lymphatic endothelia, but they also can affect distal sites. Along these lines, the lymphatic and endothelial vasculatures of the host act as a conduit for the dissemination of MC signals during inflammation. The central role of the MC-endothelial cell axis to immune homeostasis is emphasized by the fact that some of the most effective current treatments for inflammatory disorders are directed at interfering with this interaction.     

Engineering approaches to investigate the roles of lymphatics vessels in rheumatoid arthritis

Rheumatoid arthritis (RA) is one of the most common chronic inflammatory joint disorders. While our understanding of the autoimmune processes that lead to synovial degradation has improved, a majority of patients are still resistant to current treatments and require new therapeutics. An understudied and promising area for therapy involves the roles of lymphatic vessels (LVs) in RA progression, which has been observed to have a significant effect on mediating chronic inflammation. RA disease progression has been shown to correlate with dramatic changes in LV structure and interstitial fluid drainage, manifesting in the retention of distinct immune cell phenotypes within the synovium. Advances in dynamic imaging technologies have demonstrated that LVs in RA undergo an initial expansion phase of increased LVs and abnormal contractions followed by a collapsed phase of reduced lymphatic function and immune cell clearance in vivo. However, current animal models of RA fail to decouple biological and biophysical factors that might be responsible for this lymphatic dysfunction in RA, and a few attempted in vitro models of the synovium in RA have not yet included the contributions from the LVs. Various methods of replicating LVs in vitro have been developed to study lymphatic biology, but these have yet not been integrated into the RA context. This review discusses the roles of LVs in RA and the current engineering approaches to improve our understanding of lymphatic pathophysiology in RA.     

Ultrastructure of absorbing peripheral lymphatic vessel (ALPA) in guinea pig Peyer's patches

Corrosion casts from neoprene direct injection of lymphatic and blood vessels in guinea pig gut-associated lymphoid tissue, that is, solitary lymphoid follicles and Peyer's patches, have shown both their numerical density and their topographical arrangement in physiological conditions, after starvation and lymphatic stasis. The absorbing peripheral lymphatic vessel (ALPA) begins with the lacteal vessel, which continues in the mucosal lymphatic network. The latter is formed by subepithelial and interfollicular vessels wrapping single lymphoid follicles like a basket. Interfollicular vessels drain in the submucosal network, which flows into muscular tunica vessels with nonsegmentary bicuspid valves. They in turn drain lymph in subserosal precollectors and then in prelymphonodal collectors with conduction function. The follicles' germinal center and dome are completely devoid of ALPA vessels, while they are rich in blood vessels. Ultrastructurally, the ALPA vessel wall consists of a monolayer of endothelial cells devoid of pores, fenestrations, and open junctions and lacking a continuous basal lamina. Endothelial cells are joined by overlapping and interdigitating intercellular contacts, while end-to-end contacts are rare. They have a sizeable cell body, containing the nucleus and the common endocytoplasmic organelles, and a peripheral cytoplasm with actin-like filament bundles, free microvesicles or forming channels and a few rough-surfaced encloplasmic reticulum (RER) canaliculi. The presence of intraendothelial channels crossed by lymphocytes can often be detected within the endothelial wall during the different phases of cell transendothelial migration from lymphoid tissue to lymphatic vessel lumen. These channels undergo a numerical increase during starvation, while they are scarce during lymphatic stasis. We have quantitatively evaluated the prevalence of T lymphocytes in the lymph of interfollicular ALPA vessels and of prelymphonodal collectors draining the small intestine tract with or without Peyer's patches, under physiological and experimental conditions (starvation, lymphatic stasis).     

Vascular endothelial cell specification in health and disease

There are two vascular networks in mammals that coordinately function as the main supply and drainage systems of the body. The blood vasculature carries oxygen, nutrients, circulating cells, and soluble factors to and from every tissue. The lymphatic vasculature maintains interstitial fluid homeostasis, transports hematopoietic cells for immune surveillance, and absorbs fat from the gastrointestinal tract. These vascular systems consist of highly organized networks of specialized vessels including arteries, veins, capillaries, and lymphatic vessels that exhibit different structures and cellular composition enabling distinct functions. All vessels are composed of an inner layer of endothelial cells that are in direct contact with the circulating fluid; therefore, they are the first responders to circulating factors. However, endothelial cells are not homogenous; rather, they are a heterogenous population of specialized cells perfectly designed for the physiological demands of the vessel they constitute. This review provides an overview of the current knowledge of the specification of arterial, venous, capillary, and lymphatic endothelial cell identities during vascular development. We also discuss how the dysregulation of these processes can lead to vascular malformations, and therapeutic approaches that have been developed for their treatment.

     

Mast cell degranulation alters lymphatic contractile activity through action of histamine

OBJECTIVE: Mast cells reside in most tissues and in close association with blood vessels and nerves, areas where lymphatic vessels are also present. Mast cells and lymphatic vessels are two important players in the development of the inflammatory process. This study was designed to examine the effects of mast cell degranulation on the contractile activity of mesenteric lymphatic vessels. METHODS: Lymphatic vessel contractile activity was assessed in vitro by video microscopy of the mesentery of cow's milk-sensitized guinea pigs upon application of beta-lactoglobulin and compared to the response measured in sham animals. RESULTS: Application of 5-10 microM beta-lactoglobulin increased lymphatic vessel constriction frequency and decreased constriction amplitude (n = 12). This effect was not seen in sham-treated animals (n = 16) and was not due to an increased number of mast cells in the mesentery of the milk-sensitized animals, as revealed by histological examination. Two known mast cell-derived mediators, histamine and thromboxane A2, via stable mimetic U46619 also altered lymphatic pumping in a similar manner, but only pretreatment with the histamine H1 receptor antagonist pyrilamine (1 microM) could reduce the beta-lactoglobulin-induced response. The thromboxane A2 receptor antagonist, SQ 29548, and the 5-lipoxygenase inhibitor, caffeic acid, were without significant effect. CONCLUSION: In the in vitro mesenteric preparation, mast cell degranulation altered lymphatic contractile activity via the release of a mediator suggested to be histamine and the subsequent activation of H1 receptors. This action could potentially interfere with the expected ability of lymphatic vessels to reduce edema during inflammation.     

Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia

Vascular endothelial growth factor-A (VEGF-A) expression is up-regulated in several inflammatory diseases including psoriasis, delayed-type hypersensitivity (DTH) reactions, and rheumatoid arthritis. To directly characterize the biologic function of VEGF-A in inflammation, we evaluated experimental DTH reactions induced in the ear skin of transgenic mice that overexpress VEGF-A specifically in the epidermis. VEGF-A transgenic mice underwent a significantly increased inflammatory response that persisted for more than 1 month, whereas inflammation returned to baseline levels within 7 days in wild-type mice. Inflammatory lesions in VEGF-A transgenic mice closely resembled human psoriasis and were characterized by epidermal hyperplasia, impaired epidermal differentiation, and accumulation of dermal CD4+ T-lymphocytes and epidermal CD8+ lymphocytes. Surprisingly, VEGF-A also promoted lymphatic vessel proliferation and enlargement, which might contribute to the increased inflammatory response, as lymphatic vessel enlargement was also detected in human psoriatic skin lesions. Combined systemic treatment with blocking antibodies against VEGF receptor-1 (VEGFR-1) and VEGFR-2 potently inhibited inflammation and also decreased lymphatic vessel size. Together, these findings reveal a central role of VEGF-A in promoting lymphatic enlargement, vascular hyperpermeability, and leukocyte recruitment, thereby leading to persistent chronic inflammation. They also indicate that inhibition of VEGF-A bioactivity might be a new approach to anti-inflammatory therapy.